Infection with Toxoplasma gondii, even in immune competent patients, can cause severe morbidity, congenital disease, and even death. The primary means of transmission of this pathogen is through ingestion of contaminated food and water supplies, and consequently T. gondii is classified as a class B biodefense pathogen. Many questions remain about how this pathogen evades the early innate and adaptive immune response to establish persistent infection. Recent in vitro studies indicate that, like some other pathogens, T. gondii is capable of injecting proteins that alter cell function without invading the host cell. These results have led to the hypothesis that injection of proteins without invasion represents a parasite strategy to dampen host responses. Alternatively, these events may represent an aborted attempt by the parasite to invade cells and these injected (but not invaded) host populations may actually have a role in the development of protective immunity. In the proposed studies, we aim to test these competing hypotheses by tracking the functionality and fate of cells injected with proteins. Using a recently developed strain of T. gondii that secretes Cre recombinase in conjunction with its naturally injected proteins (SeCreEt parasites) combined with the Ai6 transgenic mouse strain that expresses the fluorescent protein ZsGreen1 upon Cre recombination, we will be able to identify any cell that has been injected with parasite proteins by flow cytometry and multiphoton microscopy. Indeed our preliminary studies have established that this approach allows us to distinguish infected cells and uninfected populations that have been injected with T. gondii proteins. The first aim of the study will use SeCreEt parasites and cells derived from Ai6 mice to determine the host-parasite interactions that lead to the presence of these secreted proteins in uninfected cells. This aim will utilize long-term in vito fluorescence imaging that will allow us to observe interactions between T. gondii and host immune cell populations at high resolution. From there, the second aim will examine the influence of injected parasite proteins on the functionality of immune cells in vivo. Flow cytometry analysis will be used to subset injected cell populations using ZsGreen1 reporter fluorescence and then assess the activation status of these cells and their ability to perform the effector functions required for parasite resistance. The final aim of the study will determine whether the injection of parasite proteins alters the ability of innate immune cells to prime the T cell response required for resistance to chronic T. gondii infection. Using multiphoton microscopy of live tissues, we can image ZsGreen1+ cels and quantify the interaction time between these cels and T cells specific for T. gondii antigens. Prolonged contacts between these cell populations would be indicative of T cel priming events. Together, these studies will provide valuable insights into the mechanism by which T. gondii and related pathogens evade the host immune response to establish persistent infection and pathogenesis. PUBLIC HEALTH RELEVANCE: Toxoplasma is a common opportunistic infection in patients with defects in T cell function and can cause significant disease in immune competent individuals. Though the primary site of infection is the gut, the mucosal immune response to this pathogen is not well understood. The overall goal of this proposal is to elucidate the mechanisms by which Toxoplasma suppresses the immune system to prevent host resistance.